Radioisotopes may be introduced into the human body both for purposes of imaging and for purposes of therapy. In either case, it is frequently desirable that the radioisotope be delivered to a specific location within the human body, such as a particular organ or a tumor. One radioisotope which is particularly well-suited for imaging applications is .sup.99m Tc, which has a half-life of 6 hours, a highly abundant, single .gamma.-ray with an energy of 140 keV, and low tissue deposition of ionizing radiation Rhodes, B. A. and Burchiel, S. W., In: Radioimmunoimaging and Radioimmunotherapy, Burchiel and Rhodes (Eds.), Elsevier, N.Y., 1983, p. 207!. An important objective is to get the Tc to a specific location in the body. This objective is obtained by combining the Tc with a biological molecule which will concentrate at a particular location within the body.
Currently, there are two general methods of attaching radioisotopes like Tc to biological molecules, nominally termed "direct" and "indirect" labeling Hnatowich, D. J. et al., 1993, J. Nucl. Med. 34:109-119!. In direct labeling, the radioisotope is combined directly with a biological molecule, or with a chemically reduced biological molecule. Direct labeling of proteins by .sup.99m Tc can lead to radiochemical impurities. This is undesirable for imaging purposes, because radiochemical impurities which do not concentrate at the desired biological target will lead to a loss of imaging contrast. A direct chemical pretreatment of the protein can provide sites on the biological molecule at which radioisotope can attach. For example, in the case of labeling with .sup.99m Tc, Rhodes and coworkers found that Sn.sup.+2 could directly interact with the biological molecule, e.g., a protein, to form sites on the biological molecule at which technetium ions could bind Rhodes, B. A. and Burchiel, S. W., In: Radioimmunoimaging and Radioimmunotherapy, Burchiel and Rhodes (Eds.), Elsevier, N.Y., 1983, p. 207!. However, the direct labeling approach may lead to relatively weak bonds between Tc and biological molecule, such that Tc can be lost through transchelation processes, thereby reducing radiospecificity Hnatowich, D. J. et al., 1993, J. Nucl. Med. 34:109-119!. Additionally, direct labeling protocols can alter the specificity of the biological molecule as compared to those not subjected to such protocols, thereby decreasing the amount of the labeled biological molecule actually reaching the desired target.
In the second approach to labeling, termed the "indirect" approach, one adds, to the biological molecule, an entity which will hold the radioisotope. This entity is termed a linker molecule, and must be capable of binding to both the biological molecule, frequently termed the targeting molecule, and to the radioisotope. In some applications, the biological molecule may be chemically modified to receive the linker molecule. When the linker molecule is combined to the targeting molecule, the resultant complex is termed a conjugate molecule. For example, if the biological molecule is an antibody, the resultant complex is termed an immunoconjugate molecule. When a radioisotope is subsequently combined, the resultant molecule is a radioconjugate or in the case of an antibody, a radioimmunoconjugate. Preferably, the linker molecule is conjugated to the biological molecule before the radioisotope is complexed to the linker molecule.
In the specific cases of .sup.99m Tc (used for imaging) and .sup.186 Re, .sup.188 Re, and .sup.189 Re (used for therapy), the readily available radioconjugate complexes require that the radioisotope be in a low, or chemically reduced, oxidation state. Radioisotopes of Re and Tc are typically available commercially in high oxidation states (e.g., Re.sup.+7 or Tc.sup.+7 in TcO.sub.4.sup.- Pinkerton, T. C. et al., 1985, J. Chem. Ed. 62:966-973!. Chemically reducing the radioisotope to a lower oxidation state, and maintaining that lower oxidation state prior to formation of the radioisotope/biological molecule complex is necessary. The chemical reducing agent serves to lower the oxidation state of the commercially obtained radioisotope, and a transchelator functions to maintain that lowered oxidation state prior to complexation with the linker molecule.
The commercially available form of .sup.99m Tc is pertechnetate, TcO.sub.4.sup.-, in which the technetium is in the +7 oxidation state, typically denoted Tc(VII). Operationally, the technetium is reduced to Tc(V), Tc(IV), Tc(III), or Tc(I), by the use of stannous dichloride, Sn(II)Cl.sub.2 Pinkerton, T. C. et al., 1985, J. Chem. Ed. 62:965!, by the use of stannous tartrate Rhodes, B. A. et al., In: Tumor Imaging, Burchfield and Rhodes (Eds.), Masson, N.Y. 1983, p. 111!, or by other inorganic chemical reducing agents, e.g., dithionite, borohydride, or ferrous ion in aqueous or aqueous-organic solutions at about pH 4 to about pH 7, or by other organic reducing agents Thakur, M. L. et al., 1991, Int. J. Radiat. Appl. Instrum. Part B, 18:227-233!. The reducing agent, preferably stannous ion, should be added in excess to ensure reduction of the total amount of pertechnetate present. Reduction is normally effected under an inert gas atmosphere, e.g., nitrogen or argon, at about room temperature.
In the case of directly reduced biological molecules (see below), if excess stannous ions are not present, the biological molecule/technetium complex begins to oxidize, releasing Tc as pertechnetate ions Burchfield and Rhodes, supra, at p. 113!. Furthermore, stabilizers for the stannous ion are advantageously present in the solution. It is known that ascorbate can improve specific loading of a chelator with reduced pertechnetate and minimize formation of TcO.sub.2, when the reducing agent is stannous ion. Other polycarboxylic acids, e.g., tartrate, citrate, phthalate, iminodiacetate, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and the like can also be used. Although polycarboxylic acids are mentioned, by way of illustration, any variety of anionic and/or hydroxylic oxygen-containing species could serve this function, e.g., salicylates, acetylacetonates, hydroxyacids, and the like.
While the precise role of such agents is not known, it appears that they chelate stannous ion and may prevent adventitious reactions and/or promote reduction by stabilization of stannic ions, and they may also chelate--and thereby stabilize--certain oxidation states of reduced pertechnetate, thereby serving as transchelating agents for the transfer of these technetium ions to presumably more stable chelation with one or more thiol groups and other nearby ligands on the protein PCT/US90/05196, Hansen, H. et al.!.
The chemical reduction of pertechnetate is typically carried out in the presence of a molecule which will chelate the reduced technetium, and thereby hold the reduced technetium in a lower oxidation state. Such molecule is termed a transchelator.
An example of a transchelator is tricine and its function as such is described in European Patent Application EP 0 569 132 A1. Tricine, one of Good's buffers, Biochemistry, 1966, Vol. 5, No. 2:467-477!, has been used in various applications as a buffer Calbiochem Biochemical/Immunological 1992 Catalog, BMC Biochemicals 1994 Catalog, both use tricine as a buffer for Endoproteinase Lys-C, sequencing grade! as well as a stabilizer of liquid formulations of radiolabelled compounds U.S. Pat. No. 4,390,517!. Another example of a transchelator is found in Schwartz, D. A. et al., 1991, Bioconjugate Chem. 2:333-336. Commercially available kits which will form a .sup.99m Tc-glucoheptonate complex are Glucoscan.RTM. and Gluceptate.RTM. sodium glucoheptonate kits available from DuPont Merck Pharmaceutical Co. and from Mallinckrodt, Inc., respectively.
That a transchelator can serve as a lyoprotectant to the conjugate molecule has not been described. The prior art taught that compounds such as sugars, e.g., mannitol, sucrose, and trehalose; amino acids, e.g., glycine; sugar alcohols; sugar acids; synthetic polymers and other proteins, e.g., HSA, could be used for such a purpose.
Fillers or bulking agents have often been employed in formulated aqueous protein-containing solutions where the combined concentration of other ingredients failed to allow the development of a physically robust cake. Fillers have been distinguished from other additives and employed to contribute bulk and mass to the dry product. It is understood that fillers may crystallize but that they may serve equally well where they persist in an amorphous state.
A general understanding of the physical chemistry of freezing and freeze-drying recognized a basic dichotomy in freezing behavior, all in accordance with the nature of the dissolved substances (and, to some extent, the nature of the freezing treatment). It was seen that dissolved constituents might crystallize with cooling and contribute to a eutectic behavior, or they might fail to crystallize, concentrating very highly, thereby persisting in an amorphous state and contribute to a non-eutectic state.
The understanding of the respective roles of buffer, lyoprotectant, and filler has developed in concert with the general understanding of freezing and freeze-drying behavior. Buffers, to function as such, should not crystallize during freezing or freeze-drying unless the protein is so stable that it is unaffected by the change in pH. Lyoprotectants should persist in amorphous states in order to embed protein and to prevent protein-protein interactions and other undesirable reactions. Fillers may crystallize or they may not providing that they contribute to the physical structure of the cake.
A pharmaceutical or diagnostic protein solution could be formulated with a buffer, a lyoprotectant, and a filler and the three distinct ingredients might be required. One might, for example, employ a TRIS/TRIS-HCl mixture to buffer, sucrose to lyoprotect, and mannitol to contribute cake mass. One might, on the other hand, employ a Na citrate/citric acid buffer solution to buffer, lyoprotect, and contribute cake mass. The same could be said of human serum albumin (HSA)--it is known to buffer, to protect many other proteins, and contribute mass, Na citrate buffers and HSA have each been used to fulfill these three separate needs.
Certain molecules that interact with specific targets or desired sites can be used as highly specific vehicles for the delivery of drugs or radioisotopes to target organs, tumors or thrombi in vivo. As one illustrative example, there are methods for the direct labeling of antibodies with radioisotopes, as described by Huang et al., 1980, J. Nucl. Med. 21:783 and by Rhodes, B. A. et al., In: Tumor Imaging, Burchield and Rhodes (Eds.), Masson, N.Y. (1983), p. 111. In these methods, disulfide linkages intrinsic to the antibodies are chemically reduced to generate free thiol groups, which are capable of binding radiometals such as technetium. There are two major disadvantages to the direct chemical reduction approach: (1) not all the proteins or peptides which are desirable as delivery vehicles contain readily reducible disulfide linkages and (2) chemical reduction can alter the biological activity of the reduced protein/peptide relative to the initial, unreduced protein/peptide.
Binding of the radioisotope to a linker molecule, which in turn is bound to the targeting molecule can overcome these disadvantages. The targeting molecule may be an antibody, or an antibody which has been chemically modified to facilitate binding to a linker molecule.
In the specific case of antibodies, which are glycoproteins, the linker molecule, as an example and not by way of limitation, may be attached to the carbohydrate moiety of the antibody. This may be done by first oxidizing the carbohydrate moiety to an aldehyde function. An example of addition of a linker to oxidized antibodies is provided in U.S. Pat. No. 4,741,900, incorporated herein by reference.
Various linker molecules have been proposed. The linker molecule performs two functions: (1) a given linker molecule contains functional groups which are reactive with functional groups on a given biological molecule (the targeting molecule) and (2) a given linker molecule contains functional groups which can hold a given radioisotope.
Of (1), there are linker molecules which are reactive with functional groups on proteins, especially antibodies or antibody fragments, peptides, nucleic acids or steroids. The specific functional groups of the biological molecule can include, but are not limited to, (a) oxidized carbohydrate moieties (in which case the functional group of the linker may be a primary amine, hydroxyl amine, hydrazide, thiohydrazide, phenylhydrazine, semicarbazide or thiosemicarbazide), (b) sulfhydryl (in which case the functional group of the linker may be pyridyl disulfide, haloacetate/haloacetamide or maleimide), (c) amino (in which case the functional group of the linker may be isothiocyanate, haloacetate/haloacetamide, carboxylic acid, ester or succinate) or (d) carboxylic (in which case the functional group of the linker may be an amine, hydrazide, or semicarbazide).
Of (2), the linker molecule can have functionality to hold the radioisotope to the linker molecule. In the case of a radiometal, such as .sup.99m Tc, .sup.186 Re, or .sup.188 Re, appropriately spaced C.dbd.O, C.dbd.S, or other functionality may be appropriate to hold the radioisotope to the linker molecule.
A number of bifunctional chelating agents have been reported in the scientific literature. Tolman et al. U.S. Pat. No. 4,732,864! have described the use of the cysteine rich, metal binding protein metallothonein and metallothionein fragments conjugated to targeting molecules. However, this method suffers from the fact that metallothonein is itself a large molecule and it may be difficult to purify and characterize such conjugates.
Schwartz et al. PCT WO 94/10149; Schwartz, D. A. et al., 1991, Bioconjugate Chem. 2:333-336! describe a series of bifunctional technetium chelators based on pyridyl hydrazines.
Fritzberg et al. EP 0188256; Fritzberg et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:4025! have described several examples of bifunctional dithiolate diamide technetium chelators. However, such methods for chelation of technetium are cumbersome since the compounds must be pre-chelated to technetium and then conjugated to antibodies. Also such compounds require a free thiol group for technetium chelation.
Yokoyama et al. U.S. Pat. No. 4,287,362; Arano et al., 1986, Int. J. Nucl. Med. Biol. 12:425; Yokoyama et al., 1987, J. Nucl. Med. 28:1027! describe bifunctional chelators based on thiocarbazone derivatives of 1,2 dicarbonyl compounds. These compounds have a thiocarbonyl moiety as the technetium chelating group.
EP 0 569 132 A1 discloses the use of tricine as a transchelator in a two component kit for the radiolabeling of conjugate molecules, specifically those containing 2-hydraminopyridine derivatives. This procedure required two distinct steps: (1) the combination of a solution of .sup.99m TcO.sub.4.sup.- with a lyophilized mixture of tin dichloride dihydrate and the transchelator tricine such that the Tc is reduced and chelated by tricine and (2) the combination of the resultant chemically reduced technetium solution with a solution of conjugate molecule.
For ease of storage and for ease of use, it is desirable that the components of the kit be lyophilized, which is to say, freeze-dried. It is well known to those skilled in the art that lyophilized products require a bulking agent that forms a cake and is instantly soluble upon rehydration. Typical bulking agents include sugars (e.g., mannitol, sucrose, trehalose) or amino acids (glycine). Proteins usually require a lyoprotectant during lyophilization, such that the protein is instantly soluble, does not aggregate, and retains its pharmaceutical activity upon rehydration. The issues involved in stabilizing proteins during freeze-drying have been discussed Carpenter, J. F. et al., 1991, Develop. Biol. Standard. 74:225-239; MacKenzie, A. P., 1977, Develop. Biol. Standard. 36:51-67.!
In summary, for the formation of radioconjugates, particularly those containing Tc or Re, the prior art teaches a multistep process: the radioisotope is first reduced to a lower oxidation state in the presence of a transchelator, which maintains the lowered oxidation state, and then the solution of reduced radioisotope is combined with a conjugate molecule to form a radioconjugate.
Citation or identification of any reference in the background of this application shall not be construed as an admission that such reference is available as prior art to the present invention.
There are several drawbacks with the teachings of the prior art. In multistep procedures, there is a complexity not involved in single-step procedures, such that there is a greater possibility of error. Additionally, multistep procedures take more time and may use more reagents, thus are more expensive. Further, in the compositions of the prior art there are problems associated with low radiochemical purities, with unstable reducing agents and with unstable linker molecules. Therefore, it is desirable to be able to formulate a labeling kit such that the labeling process requires only one step and radiolabeling occurs quickly, efficiently, and such that high radiochemical purities are achieved.